Monoclonal antibodies (mAbs) are a rapidly growing class of protein therapeutics. Antibody oligomers and aggregates are forms of higher-order structure of mAbs that have been linked to immunogenicity, limited stability, and reduced efficacy. Therefore, their quantitation is required by FDA for quality control. Unfortunately, to reduce volume required for therapeutic administration, mAbs are typically formulated at concentrations on the order of 100 mg/ml, which is far above the range of existing particle sizing methods. An open question has been whether dilution can lead to complex dissociation and mask higher-order structures. While sedimentation velocity analytical ultracentrifugation has become the gold standard for measuring higher-order structures in dilute solutions, new opportunities have arisen for characterizing mAb solutions much closer to formulation conditions with the new technique of nonideal sedimentation velocity (SV), recently developed in our laboratory. To explore the viability of this approach, we have embarked to determine the limits of quantitation for trace aggregates determined by nonideal SV from concentrated solutions of the NISTmAb reference protein. Different mixtures of native and heat-stressed NISTmAb have been measured by nonideal SV, and size-exclusion chromatography was carried out as an orthogonal measurement in the collaborating laboratory of Dr. John Schiel (NIST). We are currently in the data analysis stage, to evaluate the sensitivity and reliability of this approach. A second important property of mAb formulations is their colloidal stability. Low viscosity and long-term stability have been hypothesized to correlate with stronger repulsive interparticle interactions, while liquid-liquid phase separation can occur in the presence of weakly attractive interactions. These weak interparticle interactions are reflected in the particle distance distribution, which is commonly assessed by measuring second virial coefficients by static light scattering. Unfortunately, a difficulty is that light scattering is highly sensitivity to trace aggregates. To address this problem, we have resorted again to nonideal SV, taking advantage of a relationship between the second virial coefficient, the interparticle distance distribution, and the magnitude of the nonideality coefficient of sedimentation. The latter is a byproduct of nonideal SV analysis of protein solutions at high concentrations. As a result, as we have found that interparticle attractive or repulsive interactions may be measured simultaneous to the higher-order structure in nonideal SV. A third aspect of protein formulations in biotechnology are their rheological properties to make drugs easily and painlessly injectable. High viscosity is a result of mAb self-association at high concentrations. Therefore, it is desirable to measure self-association properties of mAbs in concentrated solutions, to allow the selection of clones for drug development that do not exhibit self-association. We have recently shown that nonideal SV also offers a unique potential to reveal transiently formed oligomeric structures, and to measure weak self-association of proteins. In order to evaluate this application of nonideal SV to mAb solutions, we have studied a panel of mAbs previously characterized in the published literature with regard to their self-association properties. We have found that nonideal SV can reproduce well the previous findings, and is more sensitive to self-association due to the increase in the available concentration range. Related to this project, we have embarked on the study of antibodies in serum. Although many monoclonal mAbs in biotechnological products have been found to self-associate, it has remained unclear to what extent self-association occurs among polyclonal antibodies in serum. It has also been proposed that IgG can weakly bind serum albumin. Both processes could potentially modulate IgG function through enhanced or suppressed antigen binding and Ig receptor binding. Therefore, we have undertaken efforts to further increase the concentration range of nonideal SV. Through the development of shorter optical pathlength sample holders and techniques to monitor optical aberrations, we have currently achieved an upper concentration limit that requires dilution of serum by only a factor of two. In further work we aim at closing the gap to allow the study of antibody solutions in physiological conditions.